WO2020211847A1 - Application of tegaserod in preparation of anti-tumor drugs - Google Patents

Abstract

Tegaserod or a pharmaceutically acceptable salt thereof as a JAK-STAT3 signal pathway inhibitor and an immunomodulator, and an application thereof in preparation of anti-tumor drugs. The tegaserod and the pharmaceutically acceptable salt thereof have a very good inhibitory effect on the growth of various tumor cells in vivo and in vitro, and therefore are expected to be used for treatment of various cancers.

Description

Application of Tegaserod in the Preparation of Antitumor Drugs Technical field

The invention belongs to the field of medical applications, and relates to a new medical use of tegaserod. Specifically, it is used as a JAK-STAT3 signal pathway inhibitor and immunomodulator, and can be used to prepare antitumor drugs.

Background technique

Tumors are currently the number one killer of human health and life, and current clinical drugs are far from meeting the needs of patients. The development of anti-tumor drugs is an extremely important research direction in the current drug development field.

JAK-STATs signals in cells are crucial to the conduction of cell signals and various physiological activities. Abnormal signals in this family can lead to the occurrence of many diseases, including cancer and immune-related diseases. The JAKs family includes four members: JAK1, JAK2, JAK3 and Tyk2. The STATs family downstream of JAKs contains 7 members, of which STAT3 is an important family member, which is constitutively activated by abnormal upstream tyrosine kinases in a large number of tumor cell lines and human tumors. Abnormal STAT3 signals participate in the occurrence and development of human tumors by stimulating cell proliferation, promoting angiogenesis, and inhibiting apoptosis. Therefore, inhibiting the JAK-STAT3 signaling pathway is a potentially feasible clinical treatment strategy for tumors.

The current clinical treatment strategies for inhibiting the JAK-STAT3 signaling pathway mainly include the following categories: one is tyrosine kinase inhibitors targeting STAT3 upstream signaling molecules, including JAK kinase family inhibitors; the other is blocking STAT3 gene expression or protein function , Such as through the dominant-negative STAT protein or RNAi interference for STAT3; the third is to use small molecules to inhibit STAT3 activation and dimerization.

In recent years, with the rapid increase in the development cost of new drugs and the gradual decline in the success rate of drug development, most drug patents have expired and it is difficult to follow up with new drugs. Pharmaceutical companies have developed new patents on the basis of existing drugs. Protect the drug. "New use of old drugs" has become a hot spot in international drug research and development.

Tegaserod (formula I), chemically named 2-[(5-methoxy-1H-indol-3-yl)methylene]-N-pentylcarbazide, is a selective 5- Serotonin 4 (5-HT ₄ ) receptor agonist, and its maleate was approved by the FDA in 2002 for the treatment of irritable bowel syndrome. In addition, Tegaserod has therapeutic effects on various gastrointestinal disorders, including heartburn, bloating, postoperative intestinal obstruction, abdominal pain and discomfort, epigastric pain, nausea, vomiting, nausea, and pseudo-intestinal infarction And gastroesophageal reflux.

Summary of the invention

The present invention unexpectedly found that tegaserod has inhibitory activity on JAK-STAT3 signaling pathway activity, and can inhibit tumor growth, and at the same time can activate the peripheral immune response and the immune response in the tumor microenvironment, and has a variety of tumors in vivo, Anti-tumor effect in vitro.

Based on the above findings, the present invention provides the application of tegaserod or a pharmaceutically acceptable salt thereof in the preparation of antitumor drugs.

As a preferred form of the present invention, the tumor is a tumor with abnormal activation of the JAK-STAT3 signaling pathway.

As a more preferred form of the present invention, the form of abnormal activation of the JAK-STAT3 signaling pathway is the increased phosphorylation level of JAK1, JAK2, JAK3, Tyk2 or STAT3, preferably, JAK1Tyr1022/1023 site, JAK2 Tyr1007/ The phosphorylation level of 1008 site, TYK2 Tyr1054/1055 site or STAT3Tyr705 site increased.

As a preferred form of the present invention, the anti-tumor drug also activates and/or enhances the immune response.

As a more preferred form of the present invention, the immune response is a mammalian immune response.

As a more preferred form of the present invention, the immune response includes the peripheral immune response and/or the immune response in the tumor microenvironment; more preferably, the activation and/or enhancement of the peripheral immune response includes leukocytes in the peripheral blood, Increase in the number of one or more of neutrophils, lymphocytes and platelets; activate and/or enhance the anti-tumor immune response in the tumor immune microenvironment, including increasing the infiltration of immune cells (CD45+) within the tumor, or partially or All increase killer T cells (CD8+), helper T cells (CD4+), activated T cells (CD4+CD69+ and CD8+CD69+), tumor infiltrating inflammatory neutrophils (CD11b+Ly6G+), monocytes/macrophages The ratio of one or more of (CD11b+Ly6C+) and natural killer cells (CD335+).

As a preferred form of the present invention, the pharmaceutically acceptable salt is tegaserod romanate.

As a preferred form of the present invention, the tumor is any of the following: brain tumor, genitourinary system tumor, lymphatic system tumor, gastric cancer, laryngeal cancer, nasopharyngeal cancer, skin cancer, bone cancer, blood cancer, leukemia, breast Cancer, histiocytic lymphoma, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, prostate cancer, cervical cancer, liver cancer, skin cancer, epithelial cell carcinoma, etc., preferably prostate cancer , Lung cancer or colon cancer.

In addition, the tegaserod or its pharmaceutically acceptable salt can also be used in combination with an anti-tumor drug currently in use or under development to increase its clinical effect.

Tegaserod and its pharmaceutically acceptable salts have very good inhibitory effects on the growth of various tumor cells in vivo and in vitro, and are expected to be used in the treatment of various cancers. The present invention provides new therapeutic candidate drugs for tumor patients, which may further improve the curative effect on patients and improve their prognosis.

Description of the drawings

Figure 1 shows the inhibitory effect of tegaserod romanate on reporter gene expression in the STAT3 luciferase drug screening system.

Figure 2 is a schematic diagram showing that tegaserod can inhibit constitutive activation and IL-6-induced STAT3 activation by immunoblotting experiments.

Figure 3 is a schematic diagram showing that tegaserod can selectively inhibit the phosphorylation of JAK kinase by immunoblotting experiments.

Figure 4 is a schematic diagram of tegaserod inhibiting tumor cell growth in vitro.

Fig. 5 is a schematic diagram showing that intraperitoneal administration of tegaserod can inhibit the growth of transplanted tumors through an A549 nude mouse transplanted tumor model.

Figure 6 Oral tegaserod can effectively inhibit the growth of non-small cell lung cancer A549 transplanted tumor in nude mice.

Figure 7 Oral tegaserod inhibits the growth of prostate cancer DU145 transplanted tumor in nude mice.

Figure 8 Oral low-dose and low-frequency tegaserod can effectively inhibit the growth of DU145 transplanted tumors in nude mice.

Figure 9 Oral tegaserod activates the immune response in the peripheral and tumor microenvironment.

Figure 10 Tegaserod activates the immune system to inhibit the growth of colorectal cancer MC38 allograft.

detailed description

The present invention provides applications of tegaserod, free forms of tegaserod, pharmaceutically acceptable salts, prodrugs, and active metabolites thereof in the preparation of antitumor drugs.

The free form of a particular salt of tegaserod can be isolated using techniques known in the art. For example, the free form can be regenerated by treating the salt with a suitable dilute aqueous alkali solution such as a dilute aqueous solution of NaOH, a dilute aqueous solution of potassium carbonate, a dilute aqueous ammonia, and a dilute aqueous solution of sodium bicarbonate. The free forms are somewhat different from their respective salt forms in certain physical properties such as solubility in polar solvents, but for the purpose of the invention, the acid and base salts are equivalent to their respective free forms in other pharmaceutical aspects.

The pharmaceutically acceptable salt of the present invention can be synthesized from tegaserod by conventional chemical methods. Generally, it is prepared by ion exchange chromatography or by reaction of a free base and a stoichiometric amount or excess of the desired salt form of an inorganic or organic acid in a suitable solvent or a combination of solvents. Therefore, the pharmaceutically acceptable salt of tegaserod of the present invention includes the conventional non-toxic salt of the compound of the present invention formed by the reaction of tegaserod and an inorganic or organic acid. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc., and also include salts derived from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, hard Fatty acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, paloxylic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetyl Salts prepared from oxymonobenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, etc., are preferably maleate.

The present invention relates to tegaserod or a pharmaceutically acceptable salt thereof as a small molecule inhibitor of JAK-STAT3 signaling pathway, and its application in the preparation of antitumor drugs.

In one embodiment, this application provides a method for treating hyperproliferative diseases or symptoms such as human or other mammalian tumors by using tegaserod or a pharmaceutically acceptable salt thereof.

In one embodiment, the compounds and their pharmaceutically acceptable salts involved in this application can be used to treat or control histocytic lymphoma, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, and pancreatic cancer , Breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, prostate cancer, nasopharyngeal cancer, epidermal cell cancer, cervical cancer, oral cancer, human fibrosarcoma, leukemia and other hyperproliferative diseases.

The tegaserod and its pharmaceutically acceptable salts involved in this application can be used to treat the following diseases and other diseases not listed below according to the following methods:

1) A method for treating breast cancer in humans or other mammals by using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ and lobular carcinoma in situ.

2) A method for treating human or other mammalian respiratory tract cancer using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to small cell, non-small cell lung cancer and bronchial adenoma and pleuropulmonary blastoma.

3) A method for treating brain cancer in humans or other mammals by using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to brainstem and subocular gliomas, cerebellar and cerebral astrocytomas, ependymomas, and neuroectoderm and pineal tumors.

4) A method for treating tumors in male and female reproductive organs of humans or other mammals by using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Tumors of male reproductive organs include but are not limited to prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer and vulvar cancer, and intrauterine tumors.

5) A method for treating tumors in the digestive tract of humans or other mammals by using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to anal cancer, colon cancer, colorectal cancer, esophageal cancer, gastric cancer, pancreatic cancer, rectal cancer, small bowel cancer or salivary gland cancer.

6) A method for treating tumors of the urethra of humans or other mammals by using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureteral cancer, bladder infiltrating papillary urothelial cancer or urethral cancer.

7) A method for treating eye cancer in humans or other mammals by using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to intraocular melanoma and retinocytoma.

8) A method for treating liver cancer in humans or other mammals by using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to hepatocellular carcinoma (stem cell carcinoma with or without fibrinoplasty), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma.

9) A method for treating human or other mammalian skin cancer by using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merck's cell skin cancer, and non-melanoma cell carcinoma.

10) A method for treating head and neck cancer in humans or other mammals by using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to throat, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, and lip and oral cavity cancer.

11) A method for treating human or other mammalian lymphoma by using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to AIDS-related lymphoma, non-Hodgkin’s lymphoma, cutaneous T-cell lymphoma, Hodgson’s disease and central nervous system lymphoma.

12) A method for treating human or other mammalian sarcoma using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, Linba sarcoma and rhabdomyosarcoma.

13) A method for treating leukemia in humans or other mammals by using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Including but not limited to acute myeloid leukemia, acute forest cell leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

According to standard pharmaceutical techniques, tegaserod or a pharmaceutically acceptable salt thereof of the present invention can be administered to mammals, preferably humans, alone or in combination with pharmaceutically acceptable receptors, excipients or diluents in pharmaceutical compositions. It can be administered orally or subcutaneously, intramuscularly, intraperitoneally, intravenously, rectal and topical, eyes, lungs, nasal cavity, and parenteral.

The active metabolites of tegaserod or its pharmaceutically acceptable salts involved in this application, and prodrugs that can be transformed into the structure of the compounds involved in this application and their pharmaceutically acceptable salts in the body are also included in this application Application claims.

The term "immune response" refers to the body's defense and recognition response to foreign components or mutated self components. According to the location of occurrence or action in the body, immune response usually includes systemic immune response and local immune response.

The term "peripheral immune response" belongs to the systemic immune response, involving the extensive activation of the immune system "place" far away from the tumor itself, including DAMP released by immune cells circulating in the bloodstream against foreign or mutated autologous or autologous cells (Danger associated Molecular Pattern) The response caused by activation, phenotypic change, or proliferation also includes the immune response in lymph nodes, spleen, or intestinal-related lymphoid tissues.

The tumor microenvironment is composed of tumor cells and tumor-infiltrating immune cells, new blood vessels and their endothelial cells, tumor-associated fibroblasts and extracellular matrix. It can promote tumor deterioration, increase tumor invasiveness, evade host immunity, and respond to treatment . The term "immune response in the tumor microenvironment" belongs to the local immune response, which usually includes the composition, activity and function of immune cells in the tumor microenvironment. In the present invention, the immune response in the tumor microenvironment includes tumor internal immune cells (CD45+), killer T cells (CD8+), helper T cells (CD4+), activated T cells (CD4+CD69+ and CD8+CD69+), and tumors Changes in the levels of infiltrating inflammatory neutrophils (CD11b+Ly6G+), monocytes/macrophages (CD11b+Ly6C+), natural killer cells (CD335+), etc. Activation and/or enhancement of the immune response in the tumor microenvironment refers to an increase in the number or proportion of all or part of the aforementioned cells.

Tegaserod can be used in combination with other drugs known to treat or improve similar conditions. When combined administration, the original drug administration method and dosage remain unchanged, while tegaserod is taken at the same time or subsequently. When tegaserod and one or more other drugs are taken at the same time, it is preferable to use a pharmaceutical composition containing one or more known drugs and tegaserod. The combination of drugs also includes taking tegaserod with one or more other known drugs in overlapping time periods. When tegaserod is used in combination with one or more other drugs, the dose of tegaserod or a known drug may be lower than when they are used alone.

The drugs or active ingredients that can be combined with tegaserod to treat tumors include but are not limited to:

Estrogen receptor modulators, androgen receptor modulators, retinal-like receptor modulators, cytotoxin/cytostatic agents, antiproliferative agents, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors Drugs, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducers, cytotoxic drugs, tyrosine protein inhibitors, EGFR inhibitors, VEGFR inhibitor, serine/threonine protein inhibitor, Bcr-Abl inhibitor, c-Kit inhibitor, Met inhibitor, Raf inhibitor, MEK inhibitor, MMP inhibitor, topoisomerase inhibitor, histamine Acid deacetylase inhibitor, proteasome inhibitor, CDK inhibitor, Bcl-2 family protein inhibitor, MDM2 family protein inhibitor, IAP family protein inhibitor, STAT family protein inhibitor, PI3K inhibitor, AKT inhibitor , Integrin blocker, interferon-α, interleukin-12, COX-2 inhibitor, p53, p53 activator, VEGF antibody, EGF antibody, etc.

In one embodiment, the drugs or active ingredients that can be combined with tegaserod to treat tumors include, but are not limited to: aldesleukin, alendronic acid, interferon, atranoin, allopurinol , Allopurinol sodium, palonosetron hydrochloride, hexamethylmelamine, aminoglumid, amifostine, amrubicin, amsidine, anatozole, dolastron, aranesp, arglabin, arsenic trioxide, arsenoxin, 5-azacytidine, azathioprine, BCG or tice BCG, betamethasone acetate, betamethasone sodium phosphate preparations, bexarotene, bleomycin sulfate, Bromourin, bortezomib, busulfan, calcitonin, alemtuzumab injection, capecitabine, carboplatin, constellation, cefesone, simo interleukin, daunorubicin, chlorambucil, cis Platinum, Cladribine, Cladribine, Chlordronate, Cyclophosphamide, Arabinoside, Dacarbazine, Actinomycin D, Daunorubicin Liposome, Dexamethasone, Dexamethasone Phosphate, Estradiol valerate, Denil Interleukin 2, Dibomet, Delorelin, Delazosan, Diethylstilbestrol, Diflucan, Docetaxel, Desoxyfluridine, Adriamycin, Dronabinol, Chin -166-Chitosan complex, eligard, labrizyme, epirubicin hydrochloride, aprepitant, epirubicin, epoetin alfa, erythropoietin, eplatin, levamisole, estrogen Glycol preparations, 17-β-estradiol, estramustine sodium phosphate, ethinyl estradiol, amifostine, hydroxyphosphoric acid, vanbifu, etoposide, fadrozole, tamoxifen preparations, filgra Stine, Finasteride, Feresti, Fluridine, Fluconazole, Fludarabine, 5-Fluorodeoxyuridine Monophosphate, 5-Fluorouracil, Fluoxymesterone, Flutamide, Formestine, 1-β-D-arabinofuranocytidine-5'-stearoyl phosphate, formustine, fulvestrant, gamma globulin, gemcitabine, gemtuzumab, mesylate Imatinib acid, carmustine glutinous rice paper capsules, goserelin, graniscilone hydrochloride, histamine, holmexine, hydrocortisone, erythro-hydroxynonyladenine, hydroxyurea, Tetan isibemozumab, idarubicin, ifosfamide, interferon alpha, interferon-alpha2, interferon alpha-2A, interferon alpha-2B, interferon alpha-n1, interferon alpha-n3 , Interferon β, Interferon γ-1a, Interleukin-2, Intron A, Iressa, Irinotecan, Catere, Lentinan Sulfate, Letrozole, Letrozole, Leuprolate Lin, Leuprolide Acetate, Levotetraimidazole, Levoleucovorin Calcium Salt, Levothyroxine Sodium, Levothyroxine Sodium Preparation, Lomustine, Clonidamine, Dronabinol, Nitrogen Mustard, Methylcobalt Amine, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogens, 6-mercaptopurine, mesna, methotrexate, methyl aminolevulinate, mitifosine , Minocycline, mitomycin C, mitotane, mitoxonquinone, trilosteine, adriamycin citrate liposome, nedaplatin, pegylated filgrastim, oprem Interleukin, neupogen, nilutamide, tamoxifen, NSC- 631570, Recombinant Human Interleukin 1-β, Octreotide, Ondansetron Hydrochloride, Dehydrocortisone Oral Solution, Oxaliplatin, Paclitaxel, Prednisone Sodium Phosphate Preparation, Peaspartase, Pirot Xing, pentostatin, streptolytic bacteria, pilocarpine hydrochloride, pirubicin, pracamycin, porfenam sodium, prednisone, stirprednisolone, prednisone, Premarin, Procarba Navel, Recombinant Human Erythropoietin, Raltitrexed, Libby, Etidronate Rhenium-186, Rituxan, Rex-A, Romotide, Pilocarpine Hydrochloride Tablets, Octreotide, Salmo Stine, Semustine, Cizonan, Sobuzosan, Sodium Methylprednisolone, Paphosic Acid, Stem Cell Therapy, Streptozocin, Strontium Chloride-89, Levothyroxine Sodium, Tamoxifen, Tamsulosin, tasonamin, tastolactone, taxotere, texitiadine, temozolomide, teniposide, testosterone propionate, methyltestosterone, thioguanine, thiotepa, thyroid stimulating hormone, tilufonate Acid, topotecan, toremifene, tositumomab, trastuzumab, trioxifan, tretinoin, methotrexate tablets, trimethylmelamine, trimethoate, acetic acid Triptorelin, Triptorelin Pamoate, Ufodine, Uridine, Valrubicin, Vesrinone, Vinblastine, Vincristine, Vinblastamide, Vinorelbine, Verulizin, Dextropropimide, Glinstatin, Sufonin, Paclitaxel protein stabilizer, acolbifene, interferon r-lb, affinitak, aminopterin, azoxifene, asoprisnil, atamestane, atraxan Tan, BAY43-9006, Avastin, CCI-779, CDC-501, Celebrex, Cetuximab, Clinato, Cyproterone Acetate, Decitabine, DN-101, Doxorubicin -MTC, dSLIM, dutasteride, edotecarin, eflornithine, exotecan, fenretinide, histamine dihydrochloride, histamine hydrogel implant, holmium-166DOTMP, Ibandronic acid, interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasoxifene, libra, lonafamib, milprexifene, minophoric acid Esters, MS-209, liposomal MTP-PE, MX-6, Nafarelin, Nemorubicin, sivarestat, Nolatrat, Olimerson, onco-TCS, osidem, paclitaxel Polyglutamate, sodium bromate, PN-401, QS-21, quasiyang, R-1549, raloxifene, leopard enzyme, 13-cistretinoin, satraplatin, theocalciferol , T-138067, tarceva, docosahexaenoic acid paclitaxel, thymosin α1, gazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, trans-MID-lo7R, vaspo Da, vaprastatide, vatalanib, verteporfin, vinflunine, Z-100 and zoledronic acid or a combination thereof.

The present invention will be further described below in conjunction with specific examples. The purpose of the provided examples is to help further understand the present invention. The specific materials, methods, etc. used therein are for describing the present invention and do not constitute a limitation on the scope of the present invention.

The experimental materials and general experimental methods used in the examples are as follows. Unless otherwise specified, they are used according to the manufacturer's instructions.

1. Antibodies and reagents

Antibodies such as p-Tyr705-STAT3, p-Tyr1022/1023-JAK1, p-Tyr1007/1008-JAK2, p-Tyr1054/1055-Tyk2 were purchased from Cell Signaling Technology, and α-Tubulin antibody was purchased from Santa Cruz. Recombinant human IL-6 cytokine was purchased from Peprotech. CD11b-PE-Cyanine 7, CD8a-PerCP-eFluor ^TM 710, CD45-APC-eFluor 780, Rat IgG1 kappa Isotype Control (eBRG1), Anti-Mo CD32/CD16 antibody were purchased from Invitrogen. CD11b-AF488, CD4-Brilliant Violet 510 ^™ , CD206-PE-Cyanine7, CD69-PE antibodies were purchased from Biolegend. Ly-6G-FITC, Ly-6C-APC antibody, red blood cell lysate, mouse tumor dissociation kit and human tumor dissociation kit were purchased from Mintianni. Fixable Viability Dye (eFluor ^TM 506) and Ki67 antibody were purchased from BD. IDEXX Procyte Dx*Reagent kit and ProCyte Dx*Stain Pack were purchased from IDEXX Small Animal Company. The p-Tyr705-STAT3 antibody was purchased from Cell Signaling Technology. Matrigel basement membrane matrix was purchased from Corning.

2. Cell culture

HeLa and SKA cells (A549 cells constructed with STAT3-luciferase reporter gene) were cultured in DMEM medium, and DU145, A549, DLD1, H460, and MC38 were cultured in RPMI 1640 medium. During routine culture, 10% fetal bovine serum (FBS), 100IU/ml penicillin and 100mg/ml streptomycin were added to the culture medium. All cells were cultured at 37°C and 5% CO ₂ environment.

3. STAT3-dependent luciferase reporter gene detection

SKA cells were seeded into a white 96-well plate at a density of 1×10 ⁴ /well, and cultured overnight at 37° C., 5% CO ₂ . Add the test drug at the concentration to be tested and act on the cells for 24 hours. Use luciferase kit (purchased from Promega) in

The luciferase activity was measured on the microplate reader.

4. Western blotting

The cells were lysed and harvested with RIPA buffer, and the lysate was separated by SDS-PAGE gel, and the protein was transferred to a nitrocellulose membrane (purchased from GE Healthcare). Add the primary antibody to the protein to be detected on the membrane, then incubate with the horseradish peroxidase-conjugated secondary antibody (purchased from Epison), and finally use Immobilon ^TM Western chemiluminescent HRP substrate (Millipore) to detect the formed immune complex , And use the Tanon5200 imaging system to take pictures and image.

5. Flow cytometry analysis of tumor cell cycle

Inoculate DU145 cells into a 6-well plate at a density of 5×10 ⁵ /well, add different concentrations of the drug to be tested or change to serum-free culture medium as required. For cell cycle measurement experiments, cells were harvested 24 hours after treatment, and stained with a cell cycle staining kit (purchased from Lianke Bio. Catalog No.: CCS012); for apoptosis measurement experiments, eBioscience ^TM Annexin V-FITC cells were treated for 48 hours Apoptosis kit (purchased from Invitrogen) staining. Finally, the stained cells were detected by flow cytometry.

6. Cell viability determination

The cells were seeded into 96-well plates at a density of 3,000 cells/well. After 18 hours, add different concentrations of the drug to be tested and the cells. After 72 hours, add 10μl resazurin (1mg/ml) to each well and incubate for another 3 hours, then use

Measure the fluorescence value at 595nm wavelength (544nm excitation wavelength) on a multifunctional microplate reader (purchased from Molecular Device).

7. Nude mice anti-tumor animal model and determination of anti-tumor activity of drugs in vivo

Female NRMI nu/nu athymic nude mice (SPF grade, 6 weeks old, weight 17-20g) were purchased from the collection organisms, and were raised in a constant temperature and humidity environment according to the standard requirements, and had a 12-hour light-dark cycle control. The animal experiment was approved by the Laboratory Animal Committee of Ocean University of China and complies with the "Guidelines for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH Publication No. 85-23, revised in 1996).

Nude mice were subcutaneously implanted with A549 cell suspension (female mice) or DU145matrigel cell suspension (male mice). After they grew to form palpable solid tumors, the model mice were randomly divided into groups, each containing 7-10 mice Mice were grouped according to the experiment for administration treatment. Throughout the experiment, the animal body weight was measured regularly. The calculation method of tumor volume is: tumor volume=0.5×length×width×width.

8. Flow cytometric analysis of immune cell surface markers

After the experiment was terminated, the mice were sacrificed with CO ₂ and an appropriate amount of tumor tissue was taken and placed in a dissociation tube containing mouse tumor dissociation reagent (MC38 transplanted tumor) or human tumor dissociated reagent (DU145 transplanted tumor), and tissue dissociation After the separated single cells are lysed and stained with dead cells, the cells are incubated with blocking solution at 4°C, the corresponding antibodies are added, and the FACSAria III flow cytometer sorter detects and analyzes the results.

9. Blood cell analysis

After the tumor-bearing mice were put to death by CO2, blood was taken from the heart, put into an EDTA anticoagulation tube, and tested for blood cell components by IDEXX ProCyte Dx automatic blood cell analyzer.

10. Immunohistochemistry

Paraffin sections were deparaffinized with xylene, washed with ethanol at all levels, antigen retrieval, and incubated in a 37°C incubator with blocking solution, and then treated with primary antibody and secondary antibody, DAB staining solution, treated with hematoxylin, differentiation solution, and ammonia. After treatment with grade ethanol and xylene, it is sealed with neutral resin.

11. Transcriptome sequencing

Take an appropriate amount of tumor tissue, quick-frozen in liquid nitrogen, and conduct eukaryotic mRNA sequencing by Mega Biotech. Based on the Illumina Novaseq 6000 sequencing platform, all the mRNAs transcribed from specific eukaryotic tissues or cells at a certain period are sequenced. The sequencing experiment uses the Illumina TruseqTM RNA sample prep kit method to build the library. Raw data is used for quality control, sequence alignment, transcript assembly, function annotation and expression analysis. DESeq2 difference analysis software was used to analyze the significant difference genes (p-value<0.05), and the KEGG signal pathway analysis was performed for the significant difference genes.

Example 1 Tegaserod exhibits inhibitory activity in a constitutive STAT3 activated luciferase cell model.

Using a luciferase expressing cell model based on constitutive STAT3 activation (see Chinese Patent 1407357), it was found that tegaserod romanate has a significant inhibitory effect on STAT3 with an IC ₅₀ of 5.06 μM (Figure 1).

Example 2 Tegaserod inhibits the activation of STAT3

Tegaserod of different concentrations was added to the normal cultured cells, and after 2 hours of action, the cell protein lysate was collected for protein immunoblotting detection, and the activation status of STAT3 was observed by the phosphorylation status of STAT3Tyr705.

The results showed that tegaserod in DU145 (Figure 2A) and A549 (Figure 2B) constitutively activated cells of STAT3 can inhibit the activation of STAT3 in a dose-dependent manner after 2 hours of drug treatment.

IL-6 is the main cytokine that activates STAT3 signaling. Hela cells were first treated with different concentrations of tegaserod for 2 hours, and then stimulated with 5ng/ml IL-6 for 10 minutes. The cells were collected and the total protein was extracted. Western detection of STAT3 phosphorylation (Y705). As shown in Figure 2C, in HeLa cells, tegaserod also inhibited the activation of STAT3 induced by IL-6 in a dose-dependent manner.

Example 3 Tegaserod inhibits JAK kinase family activity.

STAT3 is usually activated by the phosphorylation of JAK kinase upstream after autophosphorylation of JAK kinase. Different concentrations of tegaserod were added to DU145 cells. After 2 hours of incubation, it was found that tegaserod at a concentration level of 7.5μM could inhibit the phosphoric acid at JAK1 Tyr1022/1023 and JAK2 Tyr1007/1008 in DU145 cells. At a higher concentration (15μM), it also inhibits the phosphorylation of TYK2 Tyr1054/1055 (Figure 3). This indicates that tegaserod is an inhibitor of the JAK kinase family, which shows a higher affinity to JAK1 and JAK2 than to TYK2. Tegaserod specifically inhibits the activation of JAK kinase, thereby down-regulating STAT3 phosphorylation in tumor cells.

Example 4 The inhibitory effect of tegaserod on tumor cell growth.

Tegaserod has an inhibitory effect on the growth of tumor cells from many different sources (Figure 4). After 72 hours of tegaserod, the cell activity was measured to reflect the inhibitory effect of the drug on cell growth. The IC ₅₀ values of tegaserod cell growth inhibition were 2.5 μM for prostate cancer DU145 cells, 3.1 μM for lung cancer H460 cells, 5.7 μM for lung cancer A549 cells, and 8.7 μM for DLD1 colon cancer cells.

Example 5 Intraperitoneal injection of tegaserod inhibits the growth of transplanted tumors in nude mice.

In the nude mouse A549 lung cancer xenograft animal model, intraperitoneal injection of tegaserod can inhibit the growth of xenograft tumor cells in the model animal (Figure 5A and 5B). Compared with the control group, 5mg/kg tegaserod (i.p.) can effectively inhibit the growth of A549 tumor cells, and its anti-tumor effect is comparable to the 100mg/kg gefitinib (p.o.) group. During the whole experiment, the body weight of all the experimental mice did not change significantly (Figure 5C). After reaching the end of the experiment, no obvious organ damage was observed after anatomy, indicating that tegaserod has no obvious toxicity and has good drug safety. .

Example 6 Oral tegaserod can effectively inhibit the growth of non-small cell lung cancer A549 transplanted tumor in nude mice.

[Corrected according to Rule 91 29.06.2020]
Given that tegaserod is administered orally in the treatment of constipation-type irritable bowel syndrome, we tested whether oral administration of tegaserod can effectively inhibit tumor growth in the body. The results of tumor weight and tumor volume in Figure 6A and Figure 6B both show that oral tegaserod can inhibit the growth of A549 transplanted tumors in nude mice, and the data in Figure 6C shows that the mice did not lose weight at the oral dose. It shows that the dosage is safe, and no obvious drug toxicity is observed.

Example 7 Oral tegaserod inhibits the growth of prostate cancer DU145 transplanted tumor in nude mice.

[Corrected according to Rule 91 29.06.2020]
In addition, we selected the human prostate cancer cell line DU145 with constitutive activation of STAT3, and also tested the effect of tegaserod on the growth of transplanted tumors in nude mice. The results of Figures 7A and 7B show that oral tegaserod can effectively inhibit the growth of DU145 nude mice transplanted tumors, and there is no obvious drug toxicity (Figure 7C). The immunohistochemical results of phosphorylated STAT3 in Figures 7D and 7E show that tegaserod can effectively inhibit the phosphorylation level of STAT3 in DU145 nude mice transplanted tumors, and has the ability to target JAK/STAT3 signaling pathway in vivo.

Example 8 Oral low-dose and low-frequency tegaserod can effectively inhibit the growth of DU145 transplanted tumors in nude mice.

[Corrected according to Rule 91 29.06.2020]
In both A549 and DU145 nude mouse xenograft tumor models, we have observed that there is no dose dependence between the tumor inhibition rate and the dosage of tegaserod. High-dose tegaserod did not enhance its tumor suppressor activity. Next, we tried different dosing frequencies to test whether the high-dose group's dosing frequency was further reduced, and oral tegaserod could still effectively inhibit tumor growth. The results of Figures 8A and 8B both show that tegaserod has a stronger tumor suppressor ability after oral administration once every two days, and the body weight of the mice in Figure 8C shows that tegaserod at this concentration and frequency of administration There is no obvious drug toxicity. The immunohistochemical results of the cell proliferation marker Ki67 in Figs. 8D and 8E also showed that the proliferation ability of tumors decreased after tegaserod treatment at 1.25 mg/kg orally once every two days. The above results all indicate that low-dose and low-frequency oral administration of tegaserod can effectively inhibit tumor growth.

Example 9 Oral tegaserod activates the immune response in the peripheral and tumor microenvironment.

[Corrected according to Rule 91 29.06.2020]
In view of the effectiveness of tegaserod under low-dose and low-frequency administration and its concentration-independent anti-tumor activity, we speculate that tegaserod may be similar to an immunomodulator, which also modulates the tumor’s immune microenvironment in the body. The result is a dual-effect regulation that inhibits tumor growth. In order to verify this hypothesis, we first detected the changes in the composition of immune cells in peripheral blood after tegaserod administration. The blood analyzer data in Figure 9A shows that, similar to the positive drug gefitinib, tegaserod can stimulate the increase of the total number of white blood cells, neutrophils, lymphocytes and platelets in the peripheral blood, and powerfully stimulate the peripheral immune response .

[Corrected according to Rule 91 29.06.2020]
Furthermore, we detected the composition and proportion of immune cells in the transplanted tumor of DU145 nude mice by flow cytometry. The results in Figure 9B show that the administration of tegaserod can increase the proportion of immune cells (CD45+) in the tumor and increase tumor infiltration inflammation. The ratio of neutrophils (CD11b+Ly6G+), monocytes/macrophages (CD11b+Ly6C+) and natural killer cells (CD335+), thereby inhibiting the growth of tumor cells. The positive drug gefitinib also has a similar immunomodulatory effect.

[Corrected according to Rule 91 29.06.2020]
In order to further explore the immunomodulatory effect of tegaserod in the anti-tumor process, we used transcriptome sequencing technology to perform mRNA sequencing on DU145 nude mouse xenograft tumors, and obtained DU145 nude mouse xenograft tumors by comparison with the mouse reference genome The overall mRNA expression of mouse-derived cells in China. As shown in Figure 9C, there are a large number of significantly changed genes in the mouse stromal cells of the tumor samples in the tegaserod treatment group. It can be seen through the analysis of the KEGG signal pathway (Figure 9D) that the cytokine receptor interaction (cytokine -Cytokine receptor interaction and antigen processing and presentation were significantly upregulated. This result is consistent with the results of the flow cytometry analysis in Figure 9B, and both prove that tegaserod can exert an anti-tumor effect by regulating the tumor immune microenvironment.

The above results indicate that tegaserod can activate the composition and function of immune cells in the systemic and tumor microenvironment, thereby exerting a dual anti-tumor effect.

Example 10 Tegaserod activates the immune system to inhibit the growth of colorectal cancer MC38 allograft tumors.

[Corrected according to Rule 91 29.06.2020]
In the nude mouse xenograft model, we found that tegaserod has immune activation function and anti-tumor effect. Part of the acquired immune cell deficiency in nude mice has an imperfect immune system. Therefore, we further tested the anti-tumor effect and immunomodulatory effects of tegaserod in immune-sound mice. The results show that tegaserod can effectively inhibit the growth of colorectal cancer MC38 transplanted tumors (Figure 10A and 10B), and there is no obvious drug toxicity (Figure 10C). The results of flow cytometry in Figure 10D show that tegaserod promotes the infiltration of immune cells (CD45+) within the tumor. This result is consistent with the results of the DU145 nude mouse xenograft model, and both indicate the immune activation effect of tegaserod. At the same time, the infiltration of killer T cells (CD8+) and helper T cells (CD4+) in the tegaserod group increased, and the proportion of activated T cells increased (CD4+CD69+ and CD8+CD69+) (Figure 10D). These results indicate that tegaserod can activate the tumor-killing effect of acquired immune cells and further exert anti-tumor effects.

It should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the relevant conditions of the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. Application of tegaserod or its pharmaceutically acceptable salt in the preparation of antineoplastic drugs.
2. The application according to claim 1, wherein the tumor has abnormal activation of JAK-STAT3 signaling pathway.
3. The application of claim 2, wherein the abnormal activation of the JAK-STAT3 signaling pathway is in the form of increased phosphorylation levels of JAK1, JAK2, JAK3, Tyk2, or STAT3.
4. The application according to claim 3, wherein the increase in phosphorylation level is the phosphorylation level of JAK1 Tyr1022/1023, JAK2 Tyr1007/1008, TYK2 Tyr1054/1055, or STAT3 Tyr705. Promote.
5. The application according to claim 1, wherein the anti-tumor drug activates and/or enhances the immune response.
6. The use according to claim 5, wherein the immune response is a mammalian immune response.
7. The application according to claim 6, wherein the immune response includes a peripheral immune response and/or an immune response in the tumor microenvironment.
8. The use according to claim 1, wherein the pharmaceutically acceptable salt is tegaserod romanate.
9. The application according to any one of claims 1-8, wherein the tumor is a brain tumor, genitourinary system tumor, lymphatic system tumor, gastric cancer, laryngeal cancer, nasopharyngeal cancer, skin cancer, bone cancer, blood cancer, Leukemia, breast cancer, histiocytic lymphoma, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, prostate cancer, cervical cancer, liver cancer, skin cancer, or epithelial cell cancer.
10. The application according to claim 9, wherein the tumor is prostate cancer, lung cancer or colon cancer.

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